This project encompasses approaches to understand how current anti-tubercular chemotherapy works using the most modern technologies and to develop new and improved therapies and therapeutic approaches. Individual projects within this framework are (1) developing structural and functional imaging techniques using PET/CT for use in live, M. tuberculosis (Mtb) infected animals, (2) development of advanced animal models for predicting drug efficacy under conditions that exactly mimic those experienced by TB patients, (3) understanding the activity of various drugs in animal models of tuberculosis therapy, (4) correlating responses seen in animal models with the pathology and response to therapy observed in human TB, and (5) developing techniques for assessing drug distribution, penetration, and pharmacokinetics in vivo. Most of our PET/CT studies have used 18F-2-fluoro-2-deoxyglucose (FDG) to image the metabolism of the eukaryotic cells in TB lesions in our animal models of tuberculosis. We are also identifying small molecules that could be used to specifically and endogenously label Mtb in vivo to be used as PET radiotracers. We are focusing MTb antigen 85 enzymes that are expressed on the exterior of MTbs cell wall and can incorporate exogenous trehalose (a nonmammalian disaccharide consisting of a two 1-1 ,-linked glucose monomers) as either the mono- or dimycolate in the cell wall. We used these enzymes to chemically incorporate 18F trehalose (FDT) into bacteria in the lesions of infected rabbits and marmosets. FDT PET-CT scan seems to accurately reflect low and high bacterial burden in marmoset lesions assayed for bacterial load. This is a promising sign that the FDT will be able to give an earlier indication of treatment success or failure compared to FDG. We have performed a biodistribution study via PET-CT in 3 naive macaques to provide data for first in human dosing studies and are completing dose optimization, dose blocking and additional differential FDT vs FDG labeling studies in marmosets on drug therapy to support and IND application. We have continued developing a new, non-human primate (NHP) model for tuberculosis - the common marmoset. In the past we explored if the marmoset model accurately reflects the response to treatment by providing standard TB treatment (RIF, INH, PZA, and EMB) to infected symptomatic marmosets and showed the marmoset shows similar treatment results with humans including demonstrating the superior activity of standard therapy to the early regimen containing INH and streptomycin that lead to > 30% relapse rates. As a counterpart to an early bactericidal activity and paired PET/CT clinical trial we are conducting in South Africa, NexGen EBA Radiologic and Immunologic Biomarkers of Sterilizing Drug Activity in Tuberculosis; NCT02371681 we are replicating the treatment groups and observations in randomized Mtb infected marmosets. In the study, the standard regimen is deconstructed and each drug is administered by itself or in pair-wise combinations to measure the effect of the drugs on the microbiological and radiographic markers. We are looking for unique drug signatures in the radiologic features of the animals on treatment and comparing those to the histological presentation of the lesions upon necropsy. We hypothesize that understanding the specific contributions of each drug to the disease resolution will assist in the pairing of future agents into more successful and rapidly acting regimens. We continue to study another drug class, the oxazolidinone antibiotics such as linezolid which have shown significant therapeutic effects in patients with extensively drug-resistant (XDR) tuberculosis (TB) despite modest effects in rodents and no demonstrable early bactericidal activity in human phase 2 trials. These new oxazolidinones have vastly different activities in the marmoset model of tuberculosis that appear to be related to lesion type and physical distribution of the agents into the lesions. Together with scientists at Merck we have been engaged in developing novel oxazolidinones that are TB-selective and less toxic than linezolid. Throughout the reporting period we have been actively involved in testing the PK and ADME of novel candidates as well as the efficacy of advanced lead candidates with others working on this drug class. We are also studying other classes of antibiotics from partners engaged in developing these for TB through the Gates Foundation's TB Drug Accelerator program including diarylquinolines, quinolines, imidazopyridines, nitroimidazoles, and benzothiazinones among others. These classes of antibiotics are being explored as composing new regimens for treatment of MTB and understanding the specific contribution of each one to activity including consideration of spatial distribution and the kinetics of accumulation in lesions to avoid temporal and spatial black holes of monotherapy. Using non-compartmental and population pharmacokinetic approaches, we have modeled the rate and extent of distribution of isoniazid, rifampicin, pyrazinamide and moxifloxacin in rabbit tissues and human lesions in the past finding that penetration of antibiotics in necrotic tuberculosis lesions is heterogeneous and drug-specific. With each of the new drug candidate we test for in vivo efficacy with our academic and industry partners, we continue to assess the candidates penetration into granulomas and cavities in our model animals to correlate the information with any observed efficacy. In recent experiments we have tested inhibitors of QcrB, a subunit of the menaquinol cytochrome c oxidoreductase, and inhibitors active against non-replicating MTB, in both cases we found that the candidates penetrated into the central caseous region of the tubercular lesions at concentrations above the minimal inhibitory concentration (MIC) measured in vitro, but showed minimal cidal activity in vivo suggesting possible drug tolerance. Dormancy, persistence, and drug tolerance are among the factors thought to be driving the long therapy duration of tuberculosis. Assays to measure in situ drug susceptibility of Mtb bacteria in pulmonary lesions are needed to connect this gap. With colleagues, we developed an ex vivo assay to measure the cidal activity of anti-TB drugs against Mtb bacilli present in cavity caseum obtained from rabbits with active TB. We demonstrated that caseum-dwelling Mtb bacilli are largely nonreplicating, maintain viability over the course of the assay, and exhibit extreme tolerance to many first- and second-line TB drugs. Among the many drugs tested, only the rifamycins fully sterilized caseum. A trend of phenotypic drug resistance was observed in the nonreplicating in vitro models, but with notable differences: 1) caseum Mtb exhibits higher drug tolerance than observed in the Wayne and Loebel models of nonreplicating Mtb and 2) while PZA has no activity in these nonreplicating assays, it is cidal to caseum-grown Mtb (1). This finding seems to connect the long divide between demonstrating that PZA increases durable cure rates in human trials and its poor performance in other in vitro and in vivo assays. We continue to explore host-directed therapy (HDT) as a method to increase drug efficacy by increasing agent delivery to the site of infection in the rabbit model of Mtb. We have been performing a series of experiments to determine if treatment with an agent that promotes normalization of blood vessel structure such that hypoxia is decreased and drug penetration increased could improve drug access to the lesion. In recent results, Bedaquiline penetration is increased in lesions with HDT. These experiments with additional anti-tubercular agents are ongoing in the rabbit model with results monitored by FDG-PET/CT imaging, lesion histology, drug quantification and bacterial load.